Human cytomegalovirus is a significant human pathogen that establishes a life- long latent reservoir in undifferentiated cells of the myeloid lineage in part by suppressing viral immediate early (IE) lytic phase gene expression when it enters these cells. While it was known for some time that the viral tegument protein pp71 was the master regulator of IE gene expression, how this critical transcription factor actually worked remained enigmatic. In the last funding period of this grant, we determined the mechanism through which pp71 activates IE gene expression. The protein delivered from the virion tegument translocates to the nucleus of differentiated cells such as fibroblasts and degrades Daxx, a cellular transcriptional repressor and intrinsic defense protein that, prior to or in the absence of pp71 function, transcriptionally silences infecting viral genomes. By degrading Daxx, pp71 activates IE gene expression and lytic replication ensues. We further showed during the last funding period that the Daxx intrinsic defense also represses IE gene expression during the establishment of latency, and in this context is not inactivated by pp71. Daxx represses cellular gene expression in at least three ways: blocking the activity of cellular transcription factors, recruiting modifying proteins to targeted promoters, and depositing histone variant H3.3 onto non- replicating DNA. We propose in Aim 1 to decipher how each individual activity of Daxx contributes to its ability to silence viral IE gene expression at the start of both lytic and laten infections. In lytically infected fibroblasts, the Daxx intrinsic defense is quickly inactivated by pp71, but remains active in latently infected undifferentiated cells. In the last funding period we showed that Daxx is not inactivated when latency is established because tegument-delivered pp71 remains in the cytoplasm. In broad terms, this means that the entry processes into differentiated and undifferentiated cells must have at least some differences. Specifically, the subcellular localization of tegument-delivered proteins is different. In the last funding period, w used heterologous fusions between differentiated and undifferentiated cells to show that differentiated cells express a factor that permits the nuclear trafficking of tegument delivered pp71 in undifferentiated cells. We propose in Aim 2 to define the entry process HCMV uses to infect undifferentiated cells and establish latency, and to identify the factor found in differentiated cells that permits tegument-delivered pp71 access to the nucleus.